The invention concerns the genomic sequence of the FLAP gene. The invention also concerns biallelic markers of a FLAP gene and the association established between these markers and diseases involving the leukotriene pathway such as asthma. The invention provides means to determine the predisposition of individuals to diseases involving the leukotriene pathway as well as means for the diagnosis of such diseases and for the prognosis/detection of an eventual treatment response to agents acting on the leukotriene pathway.
The progression of inflammatory diseases in which the synthesis of leukotrienes plays an active role, such as asthma and arthritis, constitutes a major health problem in Western societies.
For example, the prevalence of asthma in Occidental countries has risen steadily over the last century, affecting about 10% of the population. In 1994, it afflicted more than 14 million people in the United States alone (including 4.8 million (6.9%) less than 18 year of age) whereas only 8 million people suffered from the same disease in 1982. It claims more than 5000 lives each year (including 342 deaths among persons aged less than 25 in 1993). Asthma affects one child in seven in Great Britain, and in the United States, it causes one-third of pediatric emergency-room visits. It is the most frequent chronic disease in childhood.
Bronchial asthma is a multifactorial syndrome rather than a single disease, defined as airway obstruction and characterized by inflammatory changes in the airways and bronchial hyper-responsiveness. Stimuli which cause the actual asthma attacks include allergens (in sensitized individuals), exercise (in which one stimulus may be cold air), respiratory infections and atmospheric pollutants such as sulphur dioxide. The asthmatic subject has intermittent attacks of dyspnoea (difficulty in breathing out), wheezing, and cough that can be life-threatening or even fatal.
The manifestation of asthma probably involves both genetic and environmental factors, and in most subjects the asthmatic attack consists of two phases which illustrate the pathophysiology of the condition:
an immediate phase, consisting mainly of bronchospasms due to spasms of the bronchial smooth muscle; the cells involved are mast cells releasing histamine, but also eosinophils, macrophages and platelets releasing leukotrienes, prostaglandins, and platelet-activating factor, these spasmogens added to chemotaxins and chemokins attract leukocytes into the area, setting the stage for the delayed phase;
a later phase consisting of a special type of inflammation comprising vasodilatation, oedema, mucus secretion and bronchospasm; it is caused by inflammatory mediators released from activated cytokine-releasing T cells and eosinophils, and, possibly, neuropeptides released by axon reflexes; these mediators cause damage and loss of bronchial epithelium.
The strongest identifiable predisposing factor for developing asthma is atopy, the predisposition for the development of an IgE-mediated response to common aeroallergens. When IgE binds to the IgE receptors on the cells, the system becomes primed so that subsequent re-exposure to the relevant allergen will cause an asthmatic attack. Most asthma cases (95%) are associated with atopy.
Further to their above-mentioned role in asthma, leukotrienes are more generally involved in host defense reactions and play an important role in immediate hypersensitivity as well as in inflammatory diseases other than asthma such as inflammatory bowel disease, psoriasis and arthritis.
The Leukotriene Pathway
Leukotrienes are products of the Lipoxygenase pathways. Lipoxygenases are soluble enzymes located in the cytosol and are found in lung, platelets, mast cells, and white blood cells. The main enzyme in this group is 5-Lipoxygenase which is the first enzyme in the biosynthesis of leukotrienes.
The first step in leukotriene biosynthesis is the release of arachidonic acid from membrane phospholipids upon cell stimulation (for example, by immune complexes and calcium ionophores). Arachidonic acid is then converted into leukotrienes A4 by a 5-Lipoxygenase (5-LO) which translocates to the cell membrane where it becomes associated to a protein called xe2x80x9cfive-Lipoxygenase activating proteinxe2x80x9d (FLAP), which is necessary for leukotriene synthesis in intact cells. 5-LO also has leukotriene A4 hydrolase activity.
Leukotriene A4 (LTA4), an unstable epoxide intermediate, is then hydrolyzed into leukotriene B4 (LTA4-hydrolase activity) or conjugated with glutathione to yield leukotriene C4 (LTC4-synthase activity) and its metabolites, leukotriene D4 and leukotriene E4. LTB4 is produced mainly by neutrophils, while cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) are mainly produced by eosinophils, mast cells, basophils, and macrophages.
LTB4 is a powerful chemotactic agent for both neutrophils and macrophages. On neutrophils, it also causes up-regulation of membrane adhesion molecules and increases the production of toxic oxygen products and the release of granule enzymes. On macrophages and lymphocytes, it stimulates proliferation and cytokine release. Thus LTB4 is an important mediator in all types of inflammations.
Cysteinyl-leukotrienes act on the respiratory and cardiovascular systems. In the respiratory system, they are potent spasmogens causing a contraction of bronchiolar muscle and an increase in mucus secretion. In the cardiovascular system, they cause vasodilatation in most vessels, but they also act as coronary vasoconstrictors. The cysteinyl-leukotrienes are of particular importance in asthma.
FLAP (5-lipoxygenase-activating Protein)
FLAP is a 18-kD membrane-bound polypeptide which specifically binds arachidonic acid and activates 5-LO by acting as an arachidonic acid transfer protein. The FLAP gene spans greater than 31 kb and consists of five small exons and four large exons (See GenBank 182657, Kennedy et al. 1991 incorporated herein by reference, Genbank M60470 for exon 1, Genbank M63259 for exon 2, Genbank M63260 for exon 3, Genbank M63261 for exon 4, and Genbank M6322 for exon 5).
The nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope. Inhibitors of FLAP function prevent translocation of 5-LO from cytosol to the membrane and inhibit 5-LO activation. They are thus interesting anti-inflammatory drug candidates. Indeed, antagonists of FLAP can attenuate allergen-induced bronchoconstrictor responses which supports an important role for cysteinyl leukotrienes in mediating these asthmatic responses.
Pharmacogenomics
To assess the origins of individual variations in disease susceptibility or drug response, pharmacogenomics uses the genomic technologies to identify polymorphisms within genes that are part of biological pathways involved in disease susceptibility, etiology, and development, or more specifically in drug response pathways responsible for a drug""s efficacy, tolerance, or toxicity, including but not limited to drug metabolism cascades.
In this regard, the inflammatory phenomena which are involved in numerous diseases present a high relevance to pharmacogenomics both because they are at the core of many widespread serious diseases, and because targeting inflammation pathways to design new efficient drugs includes numerous risks of potentiating serious side-effects. The leukotriene pathway is particularly interesting since its products are powerful inflammatory molecules.
The vast majority of common diseases, such as cancer, hypertension and diabetes, are polygenic (involving several genes). In addition, these diseases are modulated by environmental factors such as pollutants, chemicals and diet. This is why many diseases are called multifactorial; they result from a synergistic combination of factors, both genetic and environmental.
For example, in addition to the evidenced impact of environmental factors on the development of asthma, patterns of clustering and segregation analyzes in asthmatic families have suggested a genetic component to asthma. However, the lack of a defined and specific asthma phenotype is proving to be a major hurdle for reliably detecting asthma-associated genes.
Asthma is usually diagnosed through clinical examination and biological testing. The non-specific bronchial hyper-responsiveness that accompanies asthma is measured by the variation of airflow triggered in a patient by the administration of a bronchoconstrictor such as histamine or methacholine. Atopy is detected by skin prick tests that measure serum IgE titers. Standard symptom questionnaires are also commonly used to detect symptoms characteristic of, but not unique to, asthma (like nocturnal wheeze and breathlessness).
However, there is no straightforward physiological or biological blood test for the asthmatic state. Despite advances in understanding the pathophysiology of asthma and its development, evidence suggests that the prevalence of the asthmatic state and the severity of asthma attacks is underestimated. As a result, adequate asthma treatment is often delayed, thereby allowing the inflammation process to better establish itself. Thus, there is a need for an efficient and reliable asthma diagnostic test.
Drug efficacy and toxicity may also be considered as multifactorial traits that involve genetic components in much the same way as complex diseases. In this respect, there are three main categories of genes that may theoretically be expected to be associated with drug response, namely genes linked with the targeted disease, genes related to the drug""s mode of action, and genes involved in the drug""s metabolism.
The primary goal of pharmacogenomics in the study of asthma is to look for genes that are related to drug response. It can first provide tools to refine the design of drug development by decreasing the incidence of adverse events in drug tolerance studies, by better defining patient subpopulations of responders and non-responders in efficacy studies and, by combining the results obtained therefrom, to further allow for better individualized drug usage based on efficacy/tolerance prognosis.
Pharmacogenomics can also provide tools to identify new targets for drug design and to optimize the use of already existing drugs, in order to either increase their response rate and/or exclude non-responders from particular treatments, or decrease undesirable side-effects and/or exclude from corresponding treatment patients with significant risk of undesirable side-effects.
For this second application of pharmacogenomics, the leukotrienes pathway is also useful because many anti-asthmatic and anti-inflammatory agents which act through the leukotrienes pathway are under development, most of which show some incidence of severe side-effects.
For example, there are two major categories of anti-asthma drugs: bronchodilators and anti-inflammatory agents. Bronchodilators are effective in reversing the bronchospasm of the immediate phase of the disease. Drugs used as bronchodilators include the xcex22-adrenoceptor agonists (dilating the bronchi by a direct action on the smooth muscle, e.g. salbutamol), the xanthines (e.g. theophylline) and the muscarinic-receptor antagonists (e.g. ipratropium bromide). These represent the short term attack symptomatic treatment.
Anti-inflammatory agents are effective in inhibiting or preventing the production of inflammatory components in both asthma phases. They include glucocorticoids, sodium cromoglycate and histamine H1-receptor antagonists. These agents represent the current long term treatment of the asthmatic state.
However, none of these currently used anti-asthmatic drugs is completely satisfactory as none actually xe2x80x9ccuresxe2x80x9d all patients with the disease. Glucocorticoids are the most interesting active compounds in this regard but they have potentially serious unwanted side-effects (oropharyngeal candidiasis, dysphonia and osteoporosis for inhaled glucocorticoids, and mood disturbances, increased appetite and loss of glucose control in diabetics for systemic glucocorticoids).
In recent years, more effective and selective leukotriene biosynthesis inhibitors (e.g., 5-LO and FLAP-binding inhibitors) have been developed and used as novel therapies for bronchial asthma and other inflammatory disorders. For example, Zileuton (Zyflo(copyright)), an inhibitor of 5-LO commercialized by Abbott Laboratories (Abbott Park, Ill.), has been shown to improve airway function and to reduce asthma-related symptoms.
Unfortunately, undesirable side-effects such as acute exacerbation of asthma, dyspepsia and elevated liver enzymes have been reported in clinical trials for Zileuton. There is also concern about drug interactions with hepatically cleared medicaments.
Thus, in addition to the need for the development of an efficient and reliable asthma diagnostic test, there is also a need to develop more effective and better targeted therapeutic strategies acting on the leukotrienes pathway with reduced side-effects and low toxicity for the user. One way to achieve this in the relative short term would be through the use of pharmacogenomics results, to better define the use of existing drugs or drug candidates in order to enhance the benefit/risk ratio on target subpopulations of patients.
The present invention stems from the isolation and characterization of the whole genomic sequence of the FLAP gene including its regulatory regions. Oligonucleotide probes and primers hybridizing specifically with a genomic sequence of FLAP are also part of the invention. A further object of the invention consists of recombinant vectors comprising any of the nucleic acid sequences described in the present invention, and in particular recombinant vectors comprising the regulatory region of FLAP or a sequence encoding the FLAP enzyme, as well as cell hosts comprising said nucleic acid sequences or recombinant vectors. The invention also encompasses methods of screening of molecules which modulate or inhibit the expression of the FLAP gene. The invention also comprises a new allelic variant of the FLAP protein.
The invention is also directed to biallelic markers that are located within the FLAP genomic sequence, these biallelic markers representing useful tools in order to identify a statistically significant association between specific alleles of the FLAP gene and diseases involving the leukotriene pathway such as inflammatory diseases, or between specific alleles of FLAP gene and either side-effects resulting from the administration of agents acting on the leukotriene pathway, preferably Zileuton, or a beneficial response to treatment with agents acting on the leukotriene pathway. These associations are within the scope of the invention.
More particularly, the present invention stems from the identification of genetic associations between alleles of biallelic markers of the FLAP gene and asthma, as confirmed and characterized in a panel of human subjects.
Methods and products are provided for the molecular detection of a genetic susceptibility in humans to diseases involving the leukotriene pathway such as inflammatory diseases and comprising, among others, asthma, arthritis, psoriasis and inflammatory bowel disease. They can be used for diagnosis, staging, prognosis, and monitoring of such diseases, which processes can be further included within treatment approaches. The invention also provides for the efficient design and evaluation of suitable therapeutic solutions including individualized strategies for optimizing drug usage, and screening of potential new medicament candidates.